JP5982810B2 - Kit containing test piece - Google Patents

Description

  The present invention relates to a kit including a test piece.

  Test strips for detecting drug susceptibility in Mycobacterium tuberculosis (Patent Documents 1 and 2), test strips for determining the drug resistance of Mycobacterium tuberculosis (Patent Document 3), detection of Mycobacterium tuberculosis and non-tuberculous mycobacteria Test specimens (Patent Document 4) and the like have been reported. These test pieces are all means for quickly and easily determining drug sensitivity etc. by detecting the presence or absence of hybridization with the target gene. However, if the hybridization temperature is not appropriate, an incorrect determination is made. May lead to.

JP 2008-142075 A JP 2008-142076 A JP 2011-87465 A JP 2009-189283 A

  An object of this invention is to provide the kit containing the test piece for target gene detection for preventing the misjudgment produced by the hybridization temperature being improper.

  As a result of intensive studies to solve the above problems, the present inventors have fixed the hybridization temperature monitoring probe to a test piece, thereby preventing erroneous determination caused by an inappropriate hybridization temperature. The present invention has been completed by finding out what can be done.

The present invention provides a kit for detecting a target gene, the kit comprising a test piece and a standard gene, wherein the test piece hybridizes with the target gene at a temperature of T _min ° C to T _max ° C. And a first probe and a second probe for hybridization monitoring are immobilized, and the first probe hybridizes with the standard gene at a temperature lower than T _min ° C. The second probe consists of an oligonucleotide that does not hybridize with the standard gene at a temperature of T _min ° C or higher, and the second probe hybridizes with the standard gene at a temperature of T _max ° C or lower, but at a temperature higher than T _max ° C It consists of an oligonucleotide that does not hybridize to a standard gene. The temperature range from T _min ° C to T _max ° C is a temperature range in which the detection probe can appropriately hybridize with the target gene.

  In one embodiment, either the 5 'or 3' end of the standard gene is modified with a radioisotope, fluorescent material, chemiluminescent material or biotin.

  In one embodiment, the kit further comprises a gene denaturing reagent containing the standard gene.

  The present invention also provides a method for detecting a target gene, in which the kit is used, (1) the first probe hybridizes with the standard gene, and the second probe is the standard. If it does not hybridize with a gene, it is determined that the hybridization temperature between the detection probe and the target gene is appropriate. (2) Both the first probe and the second probe hybridize with the standard gene. When soybeans are used, or when both the first probe and the second probe do not hybridize with the standard gene, it is determined that the hybridization temperature between the detection probe and the target gene is not appropriate.

  ADVANTAGE OF THE INVENTION According to this invention, the kit containing the test piece for target gene detection for preventing the misjudgment produced by the hybridization temperature being improper can be provided.

It is the schematic which shows the test piece of this invention typically. It is a figure which illustrates the base sequence of the standard gene of this invention and the oligonucleotide of the probe for a hybridization monitor, and the hybridization result.

  The present invention provides a kit for detecting a target gene, the kit comprising a test strip and a standard gene.

  The target gene is not particularly limited, and examples thereof include a gene for identifying a microorganism, a gene related to drug sensitivity or resistance of a microorganism, and a wild type or mutant gene.

(Test pieces)
On the test piece, a detection probe composed of an oligonucleotide that hybridizes with a target gene at a predetermined temperature (T ° C.), and a first probe and a second probe for hybridization monitoring are fixed at different positions. Preferably, each probe is fixed at regular intervals. Furthermore, a control or marker for confirming color development may be fixed.

  The test piece can be prepared by physically or chemically immobilizing each probe on the support surface. The carrier is not particularly limited, and examples thereof include organic materials such as vinyl polymer, nylon, polyester, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, and nitrocellulose; inorganic materials such as glass and silica; metallic materials such as gold and silver. Etc. An organic material is preferable in terms of easy molding processability, and polyesters such as polyethylene terephthalate are more preferable. These carriers are preferably white in order to make the color development easily visible in the detection by the color development method.

  Various known methods can be used as a method for physically immobilizing the probe on the surface of the carrier. For example, there is a method of coating the carrier surface with a polycationic polymer such as polylysine. According to this method, the efficiency of immobilization can be increased by electrostatic interaction with a probe that is a polyanion. There is also a method of increasing the efficiency of immobilization by adding an irrelevant base sequence (such as a polythymine chain) to the end of the probe and increasing the molecular weight of the immobilized probe itself. For example, various probes can be immobilized relatively easily by discharging a solution containing a polythymine-added oligonucleotide probe from a dispenser onto a nitrocellulose membrane and irradiating with ultraviolet rays. Specifically, each probe is placed in a dispenser equipped with a 24-gauge needle, and an amount of 0.5 to 1.0 μL / min is discharged at a coating speed of 2.5 to 8.5 mm / sec. When applied on the nitrocellulose membrane at regular intervals, each probe is applied as a stripe having a width of approximately 1 to 2 mm arranged at regular intervals. The probe is fixed on the carrier by irradiating the carrier coated with the stripe of the probe with ultraviolet rays. Furthermore, if necessary, if a thin cut is made so as to cross these stripes, a large number of test pieces in which the probes are arranged in sequence can be obtained at a time.

  When the support surface is a metal material such as gold, the surface of the support is treated with a thiol having an amino group such as 2-aminoethanethiol or a disulfide compound, thereby causing electrostatic interaction with the probe that is a polyanion. It is also known that the efficiency of immobilization is improved.

  As a method for chemically immobilizing a probe by introducing a functional group, various known methods are used. For example, when the carrier material is an inorganic material such as glass or silicon, there is a method of modifying the end of the probe with a functional group capable of silane coupling reaction such as trimethoxysilane or triethoxysilane. The test piece is obtained by immersing the carrier in the solution to be contained for 24 to 48 hours, taking it out, and washing it. Alternatively, an inorganic material carrier such as glass or silicon is treated with a silane coupling agent having an amino group such as aminoethoxysilane, so that the surface of the carrier is aminated, and then a carboxylic acid-introduced probe and an amino cup are introduced. There is also a method of immobilizing the probe by ring reaction. Further, when the carrier material is a metal material such as gold or silver, the end of the probe is modified with a functional group capable of binding to a metal such as a thiol group or a disulfide group, and the carrier is added to a solution containing the modified probe. It can be fixed by immersing for ~ 48 hours, taking out and then washing.

  There is also known a method of directly synthesizing a probe having a desired sequence on the surface of a carrier using a lithography technique instead of fixing the synthesized probe.

(Detection probe)
The detection probe is a probe for detecting a target gene, and consists of an oligonucleotide that hybridizes with the target gene at a predetermined temperature (T ° C.). Preferably, the target gene consists of an oligonucleotide that does not hybridize with a gene that differs by one base at an arbitrary position. For example, it consists of an oligonucleotide having a base sequence complementary to the base sequence of the target gene. The oligonucleotide length is not particularly limited, but is preferably 10 to 30 nucleotides, more preferably 12 to 26 nucleotides. The hybridization temperature (T ° C.) is appropriately set depending on the oligonucleotide length, the nucleotide sequence of the oligonucleotide, and the like. The detection probe does not hybridize with the standard gene.

  Oligonucleotides constituting the detection probe can be designed as appropriate by using standard programs and primer analysis software such as Primer Express (Perkin Elmer), and standard oligonucleotides such as automated oligonucleotide synthesizers. Can be synthesized as appropriate using various methods.

  The detection probe is preferably modified at either the 5 'or 3' end in order to fix it to the test strip.

(Standard gene and first and second probes for hybridization monitoring)
The nucleotide length and base sequence of the oligonucleotide constituting the standard gene and the first probe and the second probe for hybridization monitoring are not particularly limited, and the detection probe for detecting the target gene is from T _min ° C to T _max ° C. The first probe hybridizes to the standard gene at a temperature lower than T _min ° C, but does not hybridize to the standard gene at a temperature of T _min ° C or higher. It consists, and the second probe, in the T _max ° C. temperature below hybridize to the standard gene, consisting of an oligonucleotide that does not the standard gene hybridize at a temperature higher than the T _max ° C.. The temperature range from T _min ° C to T _max ° C is not particularly limited as long as the detection probe can appropriately hybridize with the target gene, and is, for example, 2 to 4 ° C, preferably 2 ° C.

  The nucleotide sequence of the standard gene and the oligonucleotides constituting the first probe and the second probe for hybridization monitoring should be appropriately designed by using a standard program and primer analysis software such as Primer Express (Perkin Elmer). And can be synthesized as appropriate using standard methods such as automated oligonucleotide synthesizers.

  The first probe and the second probe for hybridization monitoring are preferably modified at either the 5 'or 3' end in order to fix to the test piece.

  The standard gene is preferably modified with a radioisotope, a fluorescent substance, a chemiluminescent substance, biotin or the like at either the 5 ′ or 3 ′ end in order to detect hybridization with the probe for hybridization monitoring. .

(kit)
The kit of the present invention includes the above test piece and the above standard gene. Furthermore, for example, a reagent for extracting gene DNA from a specimen, a gene amplification reagent, a gene denaturing reagent, a hybridization reagent, and a reagent for detecting hybridization between a probe and a gene are included. The reagent for extracting the gene DNA is not particularly limited, and examples thereof include reagents based on a phenol extraction method, a guanidine thiocinate extraction method, a vanadyl ribonucleoside complex extraction method, and the like. The gene amplification reagent includes a gene amplification primer pair, and the gene amplification primer is preferably modified with a radioisotope, a fluorescent substance, a chemiluminescent substance, biotin, or the like in order to detect hybridization between the probe and the gene. ing. The gene amplification method is not particularly limited, and examples thereof include a PCR method, a LAMP method, and an ICAN method. The reagent for gene denaturation is not particularly limited as long as the gene is denatured into single-stranded DNA. Preferably, the standard gene is included. In the hybridization reagent, the salt concentration, the surfactant, and the concentration thereof are appropriately set so that oligonucleotides having complementary base sequences hybridize. The reagent for detecting hybridization between the probe and the gene is not particularly limited. For example, when the primer for gene amplification is modified with alkaline phosphatase, nitro blue tetrazolium (NBT) / 5-bromo-4-chloro -3-Indolylphosphatase p-toluidinyl salt (BCIP) coloring reagent.

(Method for detecting target gene)
(1. Sample)
The specimen is not particularly limited, for example, body fluid such as sputum, pharyngeal swab, gastric fluid, bronchoalveolar lavage fluid, intratracheal aspirate, throat wipe and / or tissue biopsy, and culture from these And the culture obtained in this way.

(2. Extraction of gene DNA)
The method for extracting gene DNA from the specimen is not particularly limited, and examples thereof include a phenol extraction method, a guanidine thiocinate extraction method, and a vanadyl ribonucleoside complex extraction method. This step may be omitted.

(3. Target gene amplification)
The gene amplification method is not particularly limited, and examples thereof include a PCR method, a LAMP method, and an ICAN method. The gene amplification primer is preferably modified with a radioisotope, a fluorescent substance, a chemiluminescent substance, biotin or the like in order to detect hybridization between the probe and the gene. The target gene may be amplified directly from the sample without extracting the gene DNA from the sample. In order to increase the detection sensitivity, for example, when the target gene is directly amplified from the specimen, the target gene may be amplified by the Nested PCR method (Japanese Patent Publication No. 6-81600) before the PCR method. As a primer in the Nested PCR method, an oligonucleotide having a base sequence located outside the target gene is usually used as compared with the primer used in the PCR method.

(4. Detection of target gene)
The amplified target gene is denatured into single-stranded DNA and hybridized with a test piece. Conditions such as the temperature of the hybridization treatment and the time are appropriately set. As a result of the hybridization treatment, for example, when the gene amplification primer of the target gene is modified with biotin, the amplified biotin-labeled target gene becomes alkaline on the test piece when hybridized with the detection probe fixed to the test piece. By reacting phosphatase-labeled streptavidin and then reacting with NBT / BCIP, alkaline phosphatase binds to the probe on the test piece, and color develops with NBT / BCIP.

  For example, when a standard gene whose 5 ′ or 3 ′ end is modified with biotin is hybridized with a hybridization monitoring probe fixed to the test piece, the test piece is reacted with alkaline phosphatase-labeled streptavidin, and then NBT / By reacting with BCIP, alkaline phosphatase binds to the probe on the test piece, and color develops with NBT / BCIP.

  Here, when the detection probe for detecting the target gene hybridizes with the target gene at T ° C, the first probe for hybridization monitoring hybridizes with the standard gene, but the second probe for hybridization monitoring is the standard. When it does not hybridize with the gene, it is determined that the hybridization temperature between the detection probe and the target gene is appropriate, and when both the first probe for hybridization monitoring and the second probe hybridize with the standard gene, Alternatively, when neither the first probe for hybridization monitor nor the second probe hybridizes with the standard gene, it is determined that the hybridization temperature between the detection probe and the target gene is not appropriate.

  If it is determined that the hybridization temperature is not appropriate, the determination based on the result of hybridization between the detection probe and the target gene is likely to be an erroneous determination, and therefore redetection is performed.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited by these Examples.

(Example 1: Detection of RFP sensitivity using a test piece at an appropriate temperature)
(1. Preparation of test piece)
As hybridization monitoring probes, a first probe and a second probe comprising oligonucleotides having base sequences represented by the following SEQ ID NOs: 1 and 2 are prepared. Moreover, in order to detect rifampicin (RFP) sensitivity of Mycobacterium tuberculosis, probes S1 to S5 each comprising an oligonucleotide having a base sequence represented by SEQ ID NOs: 3 to 7 are prepared. Furthermore, probes R2, R4a, R4b and R5 comprising oligonucleotides having the base sequences shown in SEQ ID NOs: 8 to 11 below are prepared as probes for detecting the resistance to RFP of Mycobacterium tuberculosis. These probes are designed to hybridize with the target gene in the temperature range of 61-63 ° C. That is, T _min = 61 ° C. and T _max = 63 ° C.

First probe: 5'-atgcatggatgcatgc-3 '(SEQ ID NO: 1)
Second probe: 5'-atgcatgaatgcatgc-3 '(SEQ ID NO: 2)
Probe S1: 5'-gccagctgagccaattcat-3 '(SEQ ID NO: 3)
Probe S2: 5′-ttcatggaccagaacaacccg-3 ′ (SEQ ID NO: 4)
Probe S3: 5'-ccgctgtcggggttgacc-3 '(SEQ ID NO: 5)
Probe S4: 5′-gacccacaagcgccgact-3 ′ (SEQ ID NO: 6)
Probe S5: 5′-cgactgtcggcgctgggg-3 ′ (SEQ ID NO: 7)
Probe R2: 5′-aattcatggtccagaacaaccc-3 ′ (SEQ ID NO: 8)
Probe R4a: 5′-gttgacctacaagcgccga-3 ′ (SEQ ID NO: 9)
Probe R4b: 5′-ttgaccgacaagcgccgact-3 ′ (SEQ ID NO: 10)
Probe R5: 5′-cgactgttggcgctggg-3 ′ (SEQ ID NO: 11)

  Polythymine is added to the 5 'end of the oligonucleotide constituting each probe using terminal transferase (Promega). Specifically, 2 μL of each probe (50 mM), 0.4 μL of terminal transferase (30 unit / μL), 2 μL of thymidine triphosphate (10 pmol / μL), 2 μL of reaction buffer attached to terminal transferase, and purified water 3. 6 μL is mixed to prepare a reaction solution and reacted at 37 ° C. for 4 hours, and then 90 μL of 10 × SSC buffer solution is added to the reaction solution to obtain a polythymine-added oligonucleotide probe (1 pmol / μL). .

  Next, the polythymine-added probe is placed in a dispenser equipped with a 24-gauge needle, and discharged at a rate of 0.7 μL / min so as to form a stripe having a width of 2 mm at a coating speed of 2.5 mm / second. It is applied in a vertical direction at intervals of 2 mm on a nitrocellulose film (length 75 mm × width 150 mm: Whatman). Thereafter, the nitrocellulose membrane is irradiated with 312 nm ultraviolet rays for 2 minutes to immobilize the probe. Next, the nitrocellulose membrane is cut so as to include all the stripes, thereby producing a test piece of 5 mm × 150 mm. FIG. 1 schematically shows a test piece.

(2. Amplification of rpoB gene)
In this example, an example is shown in which a culture of Mycobacterium tuberculosis is used as a specimen, and identification of these bacteria and RFP sensitivity of Mycobacterium tuberculosis are detected. All M. tuberculosis specimens should be wild type. In order to detect RFP sensitivity, it is necessary to amplify the rpoB gene encoding the RNA polymerase β subunit that may be present in the specimen. Primer F for Mycobacterium tuberculosis comprising an oligonucleotide having the base sequence shown by SEQ ID NO: 12 below, and for Mycobacterium tuberculosis comprising an oligonucleotide having the base sequence shown by SEQ ID NO: 13 and an oligonucleotide having biotin bound to the 5 ′ end The rpoB gene in the sample is amplified by a PCR method using primer R and Taq DNA polymerase (Roche Diagnostics).

Mycobacterium tuberculosis primer F: 5'-ggatggagcgggtggtccggga-3 '(SEQ ID NO: 12)
M. tuberculosis primer R: 5'-gcacgtcgcggacctccagc-3 '(SEQ ID NO: 13)

  The PCR reaction conditions were as follows: the denaturation step at 94 ° C. for 30 seconds, the annealing step at 55 ° C. for 20 seconds and the chain extension step at 72 ° C. for 20 seconds. Amplify. Biotin is bound to the 5 'end of the amplified DNA.

(3. Detection of RFP sensitivity)
A standard gene (10 nM to 100 nM) consisting of an oligonucleotide having the base sequence shown in SEQ ID NO: 14 below and a biotin linked to the 5 ′ end in 10 μL of the amplified DNA solution, sodium hydroxide (0.4 M And 10 μL of a solution containing ethylenediaminetetraacetic acid (10 mM), well stirred and left for 5 minutes to denature the amplified DNA into single strands. To this sample solution, 1 mL of a solution containing sodium dodecyl sulfate (0.01 w / v%), sodium chloride (1.8 w / v%) and sodium citrate (1.0 w / v%) was added and stirred well. In this solution, the test piece prepared in Example 1 is shaken for 30 minutes at 62 ° C., which is an intermediate value between T _min (= 61) ° C. and T _max (= 63) ° C. Next, alkaline phosphatase-labeled streptavidin is added to the test strip and allowed to bind to biotin present at the 5 ′ end of the gene bound to each probe on the test strip. In addition, nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) are added to detect the alkaline phosphatase bound to each probe on the specimen by color development. . FIGS. 1 and 2 illustrate hybridization results.

  Standard gene: 5'-gcatgcatgcatgcatgcatgcat-3 '(SEQ ID NO: 14)

  According to this result, the area where the first probe is applied develops color, but the area where the second probe is applied does not develop color. Thereby, it can be determined that the hybridization temperature was appropriate.

(Comparative Example 1: Detection of RFP sensitivity using a test piece at a low temperature)
The same procedure as in Example 1 was performed except that the hybridization temperature in the detection of RFP sensitivity was 60 ° C., which was lower than T _min (= 61) ° C. FIGS. 1 and 2 illustrate hybridization results.

  According to this result, a color that cannot be seen at an appropriate temperature is observed in the region where the detection probe is applied, but both the region where the first probe is applied and the region where the second probe is applied are colored, It can be determined that the hybridization temperature was low.

(Comparative Example 2: Detection of RFP sensitivity using a test piece at a high temperature)
The same procedure as in Example 1 was performed except that the hybridization temperature in the detection of RFP sensitivity was set to 64 ° C., which is higher than T _max (= 63) ° C. FIGS. 1 and 2 illustrate hybridization results.

  According to this result, although the coloration seen at an appropriate temperature is not recognized in the region where the detection probe is applied (no color development or weak coloration), the region where the first probe is applied and the region where the second probe is applied Since neither color was developed, it can be determined that the hybridization temperature was high.

  ADVANTAGE OF THE INVENTION According to this invention, the kit containing the test piece for target gene detection for preventing the misjudgment produced by the hybridization temperature being improper can be provided.

Claims (4)

A kit for detecting a target gene,
The kit includes a test piece (excluding a microarray) and a standard gene,
A detection probe comprising an oligonucleotide that hybridizes with the target gene at a temperature of T _min ° C to T _max ° C, and a first probe and a second probe for hybridization monitoring are fixed to the test piece,
The first probe consists of an oligonucleotide that hybridizes with the standard gene at a temperature lower than T _min ° C, but does not hybridize with the standard gene at a temperature of T _min ° C or higher,
Second probe, although the T _max ° C. temperature below hybridize to the standard gene, consisting of an oligonucleotide that does not the standard gene hybridize at a temperature higher than the T _max ° C.,
kit.   The kit according to claim 1, wherein either the 5 'or 3' end of the standard gene is modified with a radioisotope, a fluorescent substance, a chemiluminescent substance or biotin.   Furthermore, the kit of Claim 1 or 2 containing the reagent for gene modification | denaturation containing the said standard gene. A method for detecting a target gene comprising:
Using the kit according to any one of claims 1 to 3,
(1) When the first probe hybridizes with the standard gene, but the second probe does not hybridize with the standard gene, the hybridization temperature between the detection probe and the target gene is appropriate. Judgment,
(2) When both the first probe and the second probe hybridize with the standard gene, or when both the first probe and the second probe do not hybridize with the standard gene, It is determined that the hybridization temperature between the detection probe and the target gene is not appropriate.
Method.

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